GSM7926855: Tfeb/3 SC-dKO Intact (200_4); Mus musculus; RNA-Seq
Mice were euthanized with CO2 and intact and 5DPI sciatic nerves were collected with distal nerves cut to remove ~1mm from injury site. Nerves were placed into ice cold DEPC treated PBS with RNasin where the epineurium was removed from intact nerves and then nerves were snap frozen in liquid nitrogen. Nerves were then placed into a 2ml bead mill tube with 1ml Tri-Reagent. Nerves were homogenized in a Fisherbrand Bead Mill 24 homogenizer for 30sec at 5m/s for 2 cycles. Chloroform was added and mixed and the tubes were centrifuged. The aqueous phase was removed and mixed with 70% EtOH. The mixture was then centrifuged through the NucleoSpin RNA Mini Kit (Macherey-Nagel, Cat#: 740955.50) and RNA extraction was completed following the manufacturer instructions. RNA was dissolved in RNAse free water. cDNA libraries were constructed using Ion Total RNA-Seq Kit v2 from ThermoFisher (Cat# 4479789) and manufacturers recommended protocol.